Bottom Line:
Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

ABSTRACTLa (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

Mentions:
The foregoing experiments strongly suggested that human La is targeted to the S. cerevisiae nucleus via the Kap60p/ Kap95p pathway. To address the targeting of La in human cells, we subjected recombinant hsLa–GFP to the permeabilized HeLa cell assay in the presence of recombinant human factors. As shown in Fig. 5 (first panel), the fusion protein, when added alone to permeabilized HeLa cells, did not localize to the nucleus. In the presence of HeLa cytosol, however, hsLa–GFP was rapidly and efficiently targeted to the nuclei of HeLa cells (Fig. 5, second panel). In the presence of Kapα2 and Kapβ1, a portion of hsLa–GFP could be seen to accumulate around the nuclear rim (Fig. 5, third panel). Furthermore, with the addition of Kapα2, Kapβ1, Ran and p10, the efficient nuclear import of HeLa cytosol was completely reconstituted (Fig. 5, bottom panel). As is the case with other Kapα/Kapβ1 substrates, hsLa–GFP, in the presence of either Kapα2 or Kapβ1, with or without Ran and p10, did not localize to the nucleus (data not shown). The ability to reconstitute hsLa– GFP nuclear import with human Kapα/Kapβ1 and the similarity of the previously mapped hsLa NLS to the consensus bipartite NLS, combined with our experiments in S. cerevisiae (Figs. 2 and 3) provide firm evidence that La is targeted to the nucleus via the Kapα/Kapβ1 pathway in human cells.

Mentions:
The foregoing experiments strongly suggested that human La is targeted to the S. cerevisiae nucleus via the Kap60p/ Kap95p pathway. To address the targeting of La in human cells, we subjected recombinant hsLa–GFP to the permeabilized HeLa cell assay in the presence of recombinant human factors. As shown in Fig. 5 (first panel), the fusion protein, when added alone to permeabilized HeLa cells, did not localize to the nucleus. In the presence of HeLa cytosol, however, hsLa–GFP was rapidly and efficiently targeted to the nuclei of HeLa cells (Fig. 5, second panel). In the presence of Kapα2 and Kapβ1, a portion of hsLa–GFP could be seen to accumulate around the nuclear rim (Fig. 5, third panel). Furthermore, with the addition of Kapα2, Kapβ1, Ran and p10, the efficient nuclear import of HeLa cytosol was completely reconstituted (Fig. 5, bottom panel). As is the case with other Kapα/Kapβ1 substrates, hsLa–GFP, in the presence of either Kapα2 or Kapβ1, with or without Ran and p10, did not localize to the nucleus (data not shown). The ability to reconstitute hsLa– GFP nuclear import with human Kapα/Kapβ1 and the similarity of the previously mapped hsLa NLS to the consensus bipartite NLS, combined with our experiments in S. cerevisiae (Figs. 2 and 3) provide firm evidence that La is targeted to the nucleus via the Kapα/Kapβ1 pathway in human cells.

Bottom Line:
Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La.As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins.Our results are consistent with an intermingling of the nuclear import and evolution of La.

ABSTRACTLa (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.